Rotaviruses, members of the family Reoviridae, are a major cause of severe gastroenteritis in infants and young children, causing an estimated one million deaths per year. Despite their importance, little is known about the replication cycle of these segmented, double-stranded (ds)RNA viruses. Additional information about the-molecular biology of the rotaviruses would be useful for developing vaccines and for identifying approaches to inhibit virus replication in infected individuals. Previous studies showed that the rotavirus genome is replicated asymmetrically with viral messenger (m)RNA acting as the template for minus-strand synthesis to produce dsRNA. Evidence was obtained that suggests genome replication initiates only after the packaging (assortment) of all eleven species of viral mRNAs into replication intermediates (RIs). From infected cells, three distinct types of RIs have been characterized with respect to protein composition, structure and associated replicase activity (mRNA dsRNA). The RIs are composed of structural and nonstructural proteins, and two types of RIs probably have replicase activity. Together, such studies indicated that rotavirus RNA replication and morphogenesis are concurrent processes that occur in cytoplasmic inclusions (viroplasms) in infected cells. The long-term goal of the project is to characterize rotavirus RNA replication which includes describing the assembly of proteins and mRNAs into RIs and defining the structure and function of proteins involved with the synthesis of dsRNAs. The Specific Aims of the project are as follows: (i) To characterize the structure-function of nonstructural proteins associated with RIs. In particular, the specificity, functional domains and associated activities of the RNA-binding proteins N553, N535, and NS34 will be examined. (ii) To analyze the formation of single-shelled particles and Rls. Viral proteins involved in the assembly of these intermediates will be studied using a transient vaccinia virus expression system. (iii) To characterize the structure and function of the inner capsid proteins. VP1 will be examined for RNA-binding and polymerase activity and the functional domains of VP2 and VP6 will be mapped. (iv) To analyze the conserved termini of viral mRNAs for secondary structures and recognition signals. Putative secondary structures will be examined by RNase sensitivity assay and will be tested for function. (v). To develop a rotavirus infectious RNA system. Two approaches will be used to genetically manipulate the genome of the rotaviruses, a recombinant vaccinia virus system and an exogenous mRNP system. Both systems will be used in attempts to introduce plasmid-derived mRNAs into the replication cycle of the rotaviruses.